ARTICLE
OPEN
The
FANCM:p.Arg658* truncating variant is associated with
risk of triple-negative breast cancer
Gisella Figlioli et al.
Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes
BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has
been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer
(TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for
association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2.
These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM
−/−patient-derived
immortalized
fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with
increased risk of ER-negative disease and TNBC (OR
= 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted
analysis, we con
firmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with
ER-negative breast cancer risk (OR
= 1.96; P = 0.006). The functional results indicated that all three variants were deleterious
affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we
confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC
subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the
gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors.
npj Breast Cancer (2019) 5:38 ; https://doi.org/10.1038/s41523-019-0127-5
INTRODUCTION
The genetic architecture of inherited breast cancer is complex and
involves germline pathogenic variants in high and moderate-risk
genes and polygenetic factors. The major high-penetrance breast
cancer risk genes include BRCA1 and BRCA2, which are key factors
in the DNA double-strand break repair through homologous
recombination (HR) and in the inter-strand crosslink (ICL) repair as
a part of the Fanconi Anemia (FA) pathway.
1,2Recently, based on a
prospective cohort of families carrying BRCA1 or BRCA2
patho-genic variants, the average cumulative risk by age 80 was
estimated to be 72% and 69% for carriers of BRCA1 and BRCA2
pathogenic variants, respectively.
3PALB2 has been previously
considered a moderate-risk gene, but the latest estimate of about
44% lifetime risk associated with pathogenic variants may raise
this gene to the high-risk group.
4Pathogenic variants in
moderate-penetrance genes ATM and CHEK2 are also associated
with breast cancer, conferring a 20% average lifetime risk.
5,6Recently, BARD1, RAD51D, BRIP1, and RAD51C have been proposed
as risk factors for triple-negative breast cancer (TNBC) with BARD1
and RAD51D conferring high risk, and BRIP1 and RAD51C
associated with moderate risk.
7Thus, the risk associated with
pathogenic variants in each gene may vary by breast tumor
subtype.
Many of the BRCA/FA pathway genes when altered by biallelic
mutations cause FA disease. The FANCM gene (FA
complementa-tion group M, OMIM #609644) encodes for a translocase, which is
a member of the BRCA/FA molecular pathway but has been
recently disquali
fied as a disease-causing factor for FA.
8,9Some
protein-truncating variants in the FANCM gene were described as
moderate breast cancer risk factors with a greater risk of TNBC. In
the
Finnish
population,
FANCM:c.5101 C > T
(p.Gln1701*,
rs147021911) is relatively frequent and was reported to be
associated with breast cancer with odds ratio (OR) of 1.86 with
95% con
fidence intervals (CIs) = 1.26–2.75. A larger effect was
observed in familial cases (OR
= 2.11; 95% CI = 1.43–3.32), for
estrogen receptor-negative (ER-negative) breast cancer (OR
=
2.37; 95% CI
= 1.37–4.12) and for TNBC (OR = 3.56; 95% CI =
1.81
–6.98).
10We showed an increased risk (OR
= 3.93; 95% CI =
1.28
–12.11) of the FANCM:c.5791 C > T (rs144567652) truncating
variant using familial cases and controls. In vitro analysis showed
that this variant causes the skipping of the FANCM exon 22 and
the creation of a downstream stop codon (p.Gly1906Alafs12*).
11However, in the present study we refer to the FANCM:c.5791 C > T
base change as to FANCM:p.Arg1931*, which is the conventional
amino acid annotation (consistent with the stop codon creation
according to genetic code). The FANCM:p.Arg1931* was also found
to be associated with TNBC risk in the Finnish population (OR
=
5.14; 95% CI
= 1.65–16.0).
12A burden analysis of truncating
variants discovered by a re-sequencing analysis of the entire
FANCM coding region in German cases and controls con
firmed
that FANCM pathogenic variants had a particularly high risk for
TNBC (OR
= 3.75; 95% CI = 1.0–12.85).
13To study the effect of FANCM on breast cancer risk further, we
tested three recurrent truncating variants FANCM:p.Arg658*, p.
Gln1701*, and p.Arg1931*, within the OncoArray Consortium, a
collaboration of consortia established to discover germline
genetic variants predisposing to different human cancers (e.g.,
breast, colon, lung, ovary, endometrium and prostate cancers).
14These three variants were tested for association with breast cancer
risk in 67,112 breast cancer cases, 53,766 controls, and 26,662
carriers of pathogenic variants in BRCA1 or BRCA2. We also studied
the functional effect of these three variants after their lentiviral
transduction into a FANCM
−/−patient-derived cell line in which
*email: paolo.peterlongo@ifom.eu. A full list of authors and their affiliations appears at the end of the paper.1234567
we measured survival and chromosome fragility after exposure to
diepoxybutane (DEB) or the poly (ADP-ribose) polymerase
inhibitor (PARPi) olaparib.
RESULTS
Case-control analyses
We analyzed the association of three FANCM truncating variants,
p.Arg658*, p.Gln1701*, and p.Arg1931*, with breast cancer risk for
each variant separately and using a burden analysis. We tested
67,112 invasive breast cancer cases and 53,766 controls collected
by the Breast Cancer Association Consortium (BCAC,
http://bcac.
ccge.medschl.cam.ac.uk/
) and 26,662 carriers of BRCA1 or BRCA2
pathogenic variants collected by the Consortium of Investigators
of Modi
fiers of BRCA1/2 (CIMBA,
http://cimba.ccge.medschl.cam.
ac.uk/
), of whom 13,497 were affected with breast cancer and
13,165 were unaffected.
In the BCAC dataset we assessed the breast cancer risk
associated with the FANCM variants in a primary overall analysis
and in a restricted analysis including only countries in which the
variant carrier frequencies were higher than the median of
the frequencies. In these analyses we tested association with the
variants in all available invasive breast cancer cases or in the
ER-positive, ER-negative and TNBC subgroups (Table
1
). In the overall
analysis, no evidence of association was observed, either with the
presence of any FANCM variant or with any of the three variants
individually. However, FANCM:p.Arg658* showed a higher
hetero-zygote frequency in ER-negative breast cancer cases (0.093%) than
in controls (0.035%) with a greater than two-fold increased breast
cancer risk (OR
= 2.44, 95% CI = 1.12–5.34, P = 0.034). When only
TNBC cases were considered, the association was stronger (OR
=
3.79, 95% CI
= 1.56–9.18, P = 0.009). No association with
ER-negative breast cancer or TNBC was seen for p.Gln1701* or p.
Arg1931* or for all mutations combined (Table
1
). In the
country-restricted analyses, we confirmed the association found for p.
Arg658* with risk of ER-negative disease and TNBC (OR
= 2.31,
95% CI
= 1.05–5.07, P = 0.047 and OR = 3.56, 95% CI = 1.46–8.69,
P
= 0.011, respectively). The restricted set also provided evidence
for an association between p.Arg1931* and ER-negative subgroup
(OR
= 1.96, 95% CI = 1.24–3.10, P = 0.006), though not for TNBC.
No significant association was observed for p.Gln1701* with either
subgroups (Table
1
).
Analyses of carriers of BRCA1 or BRCA2 pathogenic variants
We found no evidence of associations for FANCM:p.Arg658*, p.
Gln1701*, and p.Arg1931* truncating variants with breast cancer
risk in carriers of BRCA1 or BRCA2 pathogenic variants included in
CIMBA (Supplementary Table 1). The p.Arg658* was detected with
approximately four-fold higher frequencies in the BRCA1 affected
individuals (0.063%) in comparison to the unaffected (0.013%),
and in the BRCA2 affected individuals (0.071%) in comparison to
the unaffected (0.019%). Consistently, hazard ratios (HRs) above
two were estimated for BRCA1 (HR
= 2.4, 95% CI = 0.52–11.12) and
for BRCA2 (HR
= 2.13, 95% CI = 0.41–11.14) pathogenic variant
carriers. The frequencies of p.Gln1701* and p.Arg1931* were not
increased in affected versus unaffected individuals carrying BRCA1
or BRCA2 pathogenic variants (Supplementary Table 1).
Functional studies
We tested the functional effect of FANCM:p.Arg658*, p.Gln1701*,
and p.Arg1931* on DNA repair using genetic complementation
assays (Fig.
1
). These assays were based on the EGF280 cell line
derived from immortalized
fibroblasts from a patient who lacked
the FANCM protein due to a homozygous c.1506_1507insTA (p.
Ile503*,
rs764743944)
truncating
variant.
8Complemented
FANCM
−/−cells were tested for sensitivity to DEB and olaparib
Table 1.
Single-variant and burden analyses of FANCM:p.Arg658*, p. Gln1701* and p.Arg1931* truncating variants in overall and country-restricted invasive breast cancer cases and controlsOverall
Subgroup Carriers Non-carriers Freq % OR 95% CI P FANCM:p.Arg658* Controls 19 53,717 0.035 NA All cases 31 67,038 0.046 1.26 0.71–2.25 0.430 ER-positive 19 44,516 0.043 1.15 0.61–2.20 0.670 ER-negative 10 10,750 0.093 2.44 1.12–5.34 0.034 TNBC 7 4794 0.146 3.79 1.56–9.18 0.009 FANCM:p.Gln1701* Controls 122 53,635 0.229 NA All cases 155 66,951 0.232 1.09 0.85–1.38 0.798 ER-positive 97 44,467 0.218 1.02 0.78–1.34 0.893 ER-negative 21 10,748 0.204 0.97 0.61–1.56 0.369 TNBC 10 4794 0.229 1.09 0.57–2.10 0.149 FANCM:p.Arg1931* Controls 96 53,633 0.179 NA All cases 116 66,968 0.173 1.05 0.80–1.38 0.731 ER-positive 74 44,467 0.166 1.02 0.75–1.38 0.920 ER-negative 27 10,742 0.251 1.52 0.98–2.35 0.070 TNBC 10 4795 0.208 1.29 0.67–2.50 0.461 All variantsa Controls 237 53,455 0.443 NA All cases 302 66,736 0.452 1.02 0.86–1.21 0.823 ER-positive 190 44,323 0.427 0.96 0.79–1.16 0.698 ER-negative 58 10,700 0.548 1.23 0.92–1.64 0.154 TNBC 27 4773 0.583 1.32 0.89–1.95 0.167 Country-restricted
Subgroup Carriers Non-carriers Freq % OR 95% CI P FANCM:p.Arg658* Controls 19 48,887 0.039 NA All cases 31 59,540 0.052 1.23 0.69–2.20 0.478 ER-positive 19 39,453 0.048 1.12 0.59–2.15 0.722 ER-negative 10 9613 0.104 2.31 1.05–5.07 0.047 TNBC 7 4283 0.163 3.56 1.46–8.69 0.011 FANCM:p.Gln1701* Controls 120 48,506 0.249 NA All cases 152 58,919 0.259 1.08 0.85–1.38 0.813 ER-positive 96 38,892 0.246 1.02 0.77–1.34 0.895 ER-negative 21 9558 0.230 0.97 0.60–1.56 0.368 TNBC 10 4197 0.261 1.09 0.56–2.10 0.150 FANCM:p.Arg1931* Controls 77 34,988 0.220 NA All cases 93 37,903 0.245 1.14 0.84–1.54 0.396 ER-positive 59 25,274 0.233 1.09 0.77–1.53 0.632 ER-negative 25 5920 0.421 1.96 1.24–3.10 0.006 TNBC 10 2614 0.381 1.77 0.91–3.45 0.116 All variantsb Controls NA All cases NA ER-positive NA
2
1234567 890():,;by measuring cell survival and chromosome fragility. The FANCM
protein was not detectable in the EGF280
fibroblasts. The
transduction of these cells with lentiviral vectors carrying
wild-type (wt) FANCM cDNA and cDNAs harboring FANCM:p.Gln1701*
and p.Arg1931* variants produced, as expected, different
C-terminal truncated forms of FANCM. In the EGF280 cells
transduced with FANCM:p.Arg658* no visible band was observed
on western blot (Fig.
1
a and Supplementary Fig. 1). As we lack
information on the epitope recognized by the antibody, we could
not determine whether the p.Arg658*-derived truncated protein
was unstable or if the epitope was lost due to the truncation. We
therefore analyzed the mRNA expression of FANCM:p.Arg658* by
reverse transcription and digestion of the PCR-amplified cDNAs.
The c.1972C > T base substitution causing the p.Arg658* variant
was expected to abolish a digestion site for the restriction enzyme
TseI present in the wt sequence. TseI-digestion of wt and mutated
cDNAs clearly indicated the presence of a mutated mRNA product
in the EGF280 cells transduced with FANCM:p.Arg658* (Fig.
1
b and
Supplementary Fig. 1).
In the DEB sensitivity-based assay (Fig.
1
c), the EGF280
patient-derived cell line showed a high-sensitivity phenotype, that was
rescued by expression of the wt FANCM. EGF280 cells expressing
FANCM:p.Arg658* failed to rescue DEB sensitivity and showed
survival rates overlapping with those of the native EGF280 cells. In
comparison, cells expressing FANCM:p.Gln1701* and p.Arg1931*
variants showed an intermediate phenotype with survival rates
signi
ficantly higher than those of EGF280 cells, though
signifi-cantly lower than those of the cells expressing wt FANCM (Fig.
1
c
and Supplementary Table 2). These results were con
firmed in the
chromosome fragility tests where the number of chromatidic
breaks in cells harboring p.Gln1701* or p.Arg1931* variants was
statistically lower than that of EGF280 cells or cells expressing the
p.Arg658* and statistically higher than that of cells expressing wt
FANCM (Fig.
1
d). In the olaparib sensitivity-based assay, the
survival rates of the cell lines transduced with the three FANCM
truncating variants were not statistically different. Only at higher
olaparib concentrations (>5000 nM) the survival rates of these cell
lines were signi
ficantly lower than that of the wt FANCM cells and
higher than that of the EGF280 cells (Fig.
1
e and Supplementary
Table 3).
DISCUSSION
In this study we investigated the association of the three recurrent
FANCM truncating variants p.Arg658*, p.Gln1701*, and p.Arg1931*,
with breast cancer risk overall and by tumor subtype. While in
non-Finnish Europeans these are the three most common FANCM
truncating variants, their carrier frequency is low being 0.033, 0.21
and 0.21%, respectively (
https://gnomad.broadinstitute.org/
).
15We
conducted large case-control studies in 67,112 unselected breast
cancer cases, 53,766 controls, and 26,662 carriers of BRCA1 or
BRCA2 pathogenic variants. Furthermore, we performed functional
analyses based on a patient-derived FANCM
−/−cell line
trans-duced with vectors carrying the three FANCM variants and tested
for sensitivity to DEB or olaparib. Our genetic data suggest that
FANCM:p.Arg658* is a risk factor for ER-negative and TNBC
subtypes with statistically signi
ficant ORs of 2.44 and 3.79,
respectively. These associations were con
firmed when we
restricted the analyses to countries with higher carrier frequencies.
In these restricted analyses we also found that the p.Arg1931* was
associated with breast cancer risk in the ER-negative subtype with
statistically signi
ficant OR = 1.96. (Table
1
). These data, together
with previously published genetic studies,
10–13con
firm that
FANCM truncating variants are risk factors for breast cancer, with
a stronger association for the ER-negative and TNBC subtypes. Our
functional data, obtained in a background of a FANCM null cell
line, support these
findings showing that all three truncating
variants were deleterious; hence, it is expected that, in the
heterozygous state, any of these FANCM variants have partial
activity. In the functional tests, we also observed that olaparib had
a greater effect on survival of the cells harboring any of the
FANCM:p.Arg658*, p.Gln1701*, or p.Arg1931* variants with respect
to that on EGF280 cells complemented with wt FANCM (Fig.
1
e).
As this is consistent with previous results,
16PARP1 inhibition
might be a possible therapeutic approach to treat patients with
breast tumors associated with germline FANCM pathogenic
variants. On the contrary, the DEB sensitivity assays showed that
FANCM:p.Arg658*, is associated with a stronger impairment of
DNA repair activity, compared to p.Gln1701* and p.Arg1931*,
possibly re
flecting the position of protein truncation (Fig.
1
c, d).
FANCM encodes for a key protein of the upstream FA/BRCA
pathway mediating the assembly of the FA core complex. This
protein is 2048 AA long, possesses in its N-terminal region an
intrinsic ATP-dependent DNA translocase activity and, with its
central region, recognizes the Bloom’s complex, which is also
involved in the DNA HR repair. By interacting with its C-terminal
binding partner, the FA associated protein 24 (FAAP24), the
FANCM protein brings to sites of ICL DNA lesions the FA and the
Bloom
’s complexes initiating HR repair
17(Fig.
2
). We studied
FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* in the same
genetic FANCM
−/−background and showed that, after exposure
to DEB, the N-terminal FANCM:p.Arg658* had a statistically
stronger effect on cell survival and chromosome stability
(presumably due to less ef
ficient DNA repair activity) than did p.
Gln1701* and p.Arg1931*. This also suggests that in human living
cells the FANCM:p.Arg658* variant might impair DNA repair more
severely than p.Gln1701* and p.Arg1931*. We have shown that
in vitro both the p.Gln1701*- and the p.Arg1931*-derived FANCM
proteins are expressed and that the p.Arg658*-mRNA is
tran-scribed (Fig.
1
a, b). An N-terminus fragment including the
first 422
AA of FANCM was shown to be stable when expressed in human
cell lines,
17thus supporting the possibility that the FANCM:p.
Arg658*-derived protein may also be expressed and stable. Hence,
we hypothesize that the observed difference in survival and
chromosome fragility of cells treated with DEB may be attributable
to the diverse residual function of the different truncated forms of
FANCM. In fact, the p.Gln1701*- and the p.Arg1931*-derived forms
are expected to lose the interaction with FAAP24, but to retain the
ability of binding other FANCM interacting proteins. Hence, our
data suggest that the lack of interaction between FANCM and
FAAP24 has a less severe impact on the DNA damage response
than when protein truncation occurs upstream the FANCM
domains AA 687
–1104 and AA 1027–1362 mediating the
interaction with the FA core complex and the Bloom
’s complex,
respectively.
Previously published genetic and clinical data support our
hypothesis of a position effect. FANCM pathogenic variants were
shown to be associated with a moderate risk of developing
high-grade serous epithelial ovarian cancer, but p.Arg1931* appeared
to confer a lower risk.
18Moreover,
five female breast cancer
Table 1 continued
Country-restricted
Subgroup Carriers Non-carriers Freq % OR 95% CI P ER-negative NA
TNBC NA
In bold are indicated the statistically significant results
Freqfrequency, OR odds ratio CI confidence interval, P P-value, TNBC triple-negative breast cancer, NA not applicable
aThe burden analyses were performed by univariate logistic regression b
These analyses were not possible in the country-restricted cases and controls as different countries were included for each variant. P-values were from Pearson chi-squared test
Fig. 1 Functional studies of the FANCM:p.Arg658*, p.Gln1701* and p.Arg1931* truncating variants using the patient-derived FANCM
−/−EGF280 cell line. a Western blot showing the FANCM expression in EGF280 cells complemented with lentiviral vectors harboring the three
different variants. Bands corresponding to truncated FANCM protein were visible for EGF280
+ p.Gln1701* and p.Arg1931*, and no bands
were present for the EGF280
+ p.Arg658*. b Study of the expression of the FANCM protein in EGF280 + p.Arg658*. The c.1972C > T base
substitution, causing the p.Arg658* variant abrogates a digestion site for the restriction enzyme TseI that is present in the wild-type (wt) cDNA
sequence. Total RNA was extracted from EGF280
+ wtFANCM and from the EGF280 + p.Arg658* and subjected to reverse transcription.
PCR-ampli
fied cDNA products were digested with TseI. Digested and undigested cDNAs were loaded. In the first two lanes are shown bands of
386 bp corresponding to uncut wt cDNA, and bands of 257 and 129 bp corresponding to cut wt cDNA. In next two lanes bands of 386 bp
indicate that p.Arg658* cDNA was not cut due to the c.1972C > T base substitution abrogating the TseI site. In the two lanes after the
molecular weight marker (M) undigested and digested products of the two previous PCR products were mixed 1:1 and loaded as a control. c
Analysis of diepoxybutane (DEB) sensitivity on cell survival. The EGF280 cells expressing p.Arg658* are signi
ficantly more sensitive to DEB than
the cells expressing p.Gln1701* or p.Arg1931* (P-values from Tukey
’s range test are reported in Supplementary Table 4). EGF280 and EGF280 +
wtFANCM are used as controls (N
= 3; error bars: standard deviation). d Chromosome fragility induced by DEB treatment (100 ng/ml). Here, the
chromatidic break patterns of the cells expressing wt FANCM, of the cells harboring p.Gln1701* or p.Arg1931* variants, and of the native
EGF280 cells or the cells expressing p.Arg658* were statistically different. (P-values from chi-squared test; N
= 2). e Analysis of cellular
sensitivity to olaparib. Contrarily to what we observed in the DEB sensitivity assays, survival rates of the different complemented cell lines
were apparently not different. Human
fibroblasts (BRCA2
−/−) were homozygous for the c.469 A > T (p.Lys157*) truncating variant and were
used as a positive control. (P-values from Tukey
’s range test are reported in Supplementary Table 5; N = 3; error bars: standard deviation). All
blots derive from the same experiment and were processed in parallel
probands carrying homozygous FANCM truncating variants were
recently described.
9Three of these, two homozygous for p.
Gln1701*, and one for p.Arg1931*, developed breast cancer at age
52 years or later and their cells did not demonstrate chromosome
fragility. The other two probands were homozygous for p.Arg658*
and developed early-onset breast cancer (at age 29 and 32); in
addition, one developed several cancers, and the other
demon-strated chromosomal fragility.
9Due to the rarity of the studied mutations in most populations,
estimation of the risks is challenging. Preferably, the cases should
be examined in comparison to geographically, ethnically and
genetically matched controls. In the Finnish population, p.
Gln1701* and p.Arg1931* are reported with carrier frequency of
1.62% and of 0.92%, respectively (
https://gnomad.broadinstitute.
org/
).
15Case-control studies based on the Finnish population
showed a strong statistical evidence of association of p.Gln1701*
with ER-negative disease, with OR of 2.37 (95% CI
= 1.37–4.12, P =
0.0021), and with TNBC with ORs of 3.56 (95% CI
= 1.81–6.98, P =
0.0002),
10while p.Arg1931* was found associated with TNBC with
an OR of 5.14 (95% CI
= 1.65–16.0, P = 0.005).
12However, as our
95% CI of risk estimates for TNBC included odds ratios of 2 for
both the latter mutations, the published and our results are not
mutually exclusive. Risk estimates associated with rare variants
may depend on their frequency and the genetic background of
the population studied. Hence, pooling the data from multiple
outbred and admixed populations as it was done in the present
study, may yield different risk estimates than those derived from
geographically, ethnically and genetically matched controls, as in
the Finnish studies. Indeed, it would have been interesting to test
the FANCM variant position effect in the Finnish population, but
unfortunately the p.Arg658* is very rare if not absent in this
population (
https://gnomad.broadinstitute.org/
).
15Recent attempts to identify novel, high- to moderate-risk breast
cancer-predisposing genes have not been particularly fruitful.
However, a few genes have emerged as potential risk factors for
ER-negative disease and TNBC, with FANCM, BRIP1, and RAD51C
being among those suggested to confer moderate risk of these
subtypes. Other predisposing genes increasing the risk of
ER-negative and TNBC may also exist. Hence, further gene discovery
efforts should take into consideration that risk-associated variants
may be associated with speci
fic tumor subtypes and/or variation
in risk may depend on the variant position. In addition, we provide
evidence that lack of FANCM protein and truncating variants
identi
fied in breast cancer patients are associated with increased
sensitivity to the PARPi olaparib suggesting a therapeutic
opportunity to treat FANCM-associated breast tumors that
warrants further investigation. The PARPi sensitivity test may also
prove useful for preclinical investigation of further truncating or
missense FANCM variants.
In summary, we have shown that FANCM:p.Arg658* is
associated with risk of ER-negative breast cancer and TNBC. The
outcomes of functional assays testing the DNA repair ef
ficiency in
complemented human cells support the hypothesis that breast
cancer risk may be greater for N-terminal than C-terminal FANCM
truncating variants. Further genetic studies and meta-analyses are
warranted to derive more precise risk estimates for the different
FANCM variants.
METHODS
Study participants
The individuals included in this study were women of genetically confirmed European ancestry who were originally ascertained in 73 case-control studies from 19 countries participating in the BCAC or in 59 studies enrolling BRCA1 or BRCA2 pathogenic variants carrier from 30 countries participating in the CIMBA.
Ethics
All participating studies, listed in Supplementary Table 4 and Supplemen-tary Table 5, were approved by their ethics review boards and followed national guidelines for informed consent. However, due to the retro-spective nature of the majority of the studies, not all participant individuals have provided written informed consent to take part in the present analysis. The Milan Breast Cancer Study Group (MBCSG) was approved by ethics committee from Istituto Nazionale dei Tumori di Milano and Istituto Europeo di Oncologia, in Milan.
The BCAC studies contributed 67,112 invasive breast cancer cases and 53,766 controls. The majority of these studies were population-based, hospital-based or case-control studies nested within population-based cohorts (86%); few were family-clinic-based studies (14%; Supplementary Table 4). For each study subject, information on the disease status and the age at diagnosis or at interview were provided. Data on lifestyle risk factors were available for most subjects and clinical and pathological data were available for most cases. All these data were incorporated in the BCAC dataset (version 10). A total of 44,565 (66%) cases were ER-positive, 10,770 (16%) were ER-negative, and 4,805 (7%) were TNBC; 13,743 (20%) had a positivefirst-degree family history of breast cancer.
The CIMBA studies contributed 15,679 carriers of a pathogenic BRCA1 variant and 10,983 carriers of a pathogenic BRCA2 variant to this analysis (Supplementary Table 5). Nearly all (98%) of these carriers were ascertained through cancer genetic clinics; few carriers were recruited by population-based sampling of cases or by community recruitment. In some instances, multiple members of the same family were included. For each pathogenic variant carrier, the information on the type of the BRCA1 or BRCA2 variant, disease status, and censoring variables (see below, Statistical analyses) were collected and included in the CIMBA database.
Genotyping
Genotyping of FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* truncating variants was conducted using a custom-designed Illumina genotyping array (the “OncoArray”, Illumina, Inc. San Diego, CA, USA) at six independent laboratories. To ensure consistency of the genotype data, all laboratories used the same genotype-clusteringfile and genotyped the same set of reference-samples selected from the HapMap project. Samples with a call rate <95% and those with heterozygosity <5% or >40% were excluded. Further details of the genotype-calling and quality control have been described previously.14 The cluster plots of the three FANCM truncating variants were curated manually to confirm the automatic calls (Supplementary Fig. 2).
Statistical analyses
The BCAC data were analyzed to test the association between FANCM:p. Arg658*, p.Gln1701*, and p.Arg1931* and breast cancer risk. Logistic regression analyses were performed to estimate ORs with 95% CIs for variant carriers versus non-carriers, adjusting for country and thefirst ten principal components, as previously described.19P-values were calculated by applying the likelihood ratio test (LRT) comparing the model containing the variant carrier status as a covariate to a model without the variant carrier status. The primary analyses were performed including all invasive breast cancer cases and controls and subgrouping cases based on tumor hormonal status. We then performed a country-restricted analysis
Fig. 2 Schematic diagram of the 2,048 amino acid long FANCM protein. The functional or binding domains (BD) are indicated in black and as
reported in Deans and West, 2009. The position of the three FANCM truncating variants c.1972C > T (p.Arg658*), c.5101 C > T (p.Gln1701*) and
c.5791 C > T (p.Arg1931*) is also shown
including the 50% of the countries with the higher variant carrier frequencies. Specifically, we included only countries in which the carrier frequencies in cases and controls combined were higher than the median of the carrier frequencies observed in all countries. Median frequencies were 0.007, 0.114 and 0.163 for p.Arg658*, p.Gln1701* and p.Arg1931* carriers, respectively.
The CIMBA data were analyzed to evaluate the association between each FANCM truncating variant and breast cancer risk in carriers of BRCA1 or BRCA2 pathogenic variant. A survival analyses framework was applied. Briefly, each variant carrier was followed from the age of 18 years until the first breast cancer diagnosis, or censored as unaffected at ovarian cancer diagnosis, bilateral prophylactic mastectomy, or age at last follow-up. The analyses were performed by modelling the retrospective likelihood of the observed genotypes conditional on the disease phenotype as detailed previously.20All analyses were stratified for country. The per-allele hazard ratio (HR), 95% CIs were estimated separately for each variant. A score test was used to derive P-values for the associations. The analyses of the BCAC data were performed using STATA version 15 (StataCorp LLC, College Station, Texas, USA). The analyses of the CIMBA data were carried out using custom-written code in Python and Fortran. All statistical tests were two-sided and P-values <0.05 were considered statistically significant.
Cell lines, plasmids, and lentiviral particles production and
transduction
The immortalized patient-derived FANCM−/− cell line EGF2808 was
transduced with pLenti CMV rtTA3 Blast, a gift from E. Campeau (Addgene plasmid #26429). The doxycycline-inducible lentiviral vector pLVX-TRE3G-FANCM, a gift from N. Ameziane (Vrije Universiteit Medical Center, Amsterdam) was mutated by site-directed mutagenesis using the QuickChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) and the following PAGE purified mutagenic primers. FANCM c.1972C > T primer 1: 5’-GCCTTCTCGGAACTTGCAGTGAAAGTCATCTATCTTTTCC-3’ and primer 2: 5’-GGAAAAGATAGATGACTTTCACTGCAAGTTCCGAGAAGGC-3’ for the p.Arg658*; FANCM c.5101 C > T primer 1: 5 ’-TTAAACAATGGTCC-TATTGTTTGTTCTTCTTAACAGTGCTTGGGT-3’ and primer 2: 5’-ACCCAAG-CACTGTTAAGAAGAACAAACAATAGGACCATTGTTTAA-3’ for the p.Gln1701*. Generation of the lentiviral vector containing the FANCM:c.5791 C > T (p. Arg1931*) and transduction of the EGF280 cells were already described.11
Expression of exogenous FANCM protein was achieved supplementing cell culture medium with doxycycline (1μg/ml, final concentration). All the cell lines used in this study were routinely checked for mycoplasma contamination using the MycoAlert™ Mycoplasma Detection Kit (Lonza).
Western blot and mRNA expression studies
Cell lysis and western blot assays were performed as previously described.8 The following primary antibodies were used: mouse monoclonal anti-FANCM antibody, clone CV5.1 diluted 1:100 (ref: MABC545, MERCK Millipore), mouse monoclonal anti-Vinculin diluted 1:3000 (ref: ab18058, abcam). Western blotting detection was achieved with LuminataTM
Classico (Millipore) (Vinculin) and LuminataForteTM (Millipore) (FANCM). We used RT PCR to test the expression of the mutant FANCM:p.Arg658*. Total RNA was extracted (RNeasy Mini Kit Qiagen) from the wtFANCM and FANCM:p.Arg658* transduced EGF280 cell lines. Reverse transcription was performed using High-Capacity RNA-to-cDNA Kit (Thermofisher); a cDNA region corresponding to the FANCM sequence containing the amino acid (AA) position Arg658 was amplified by PCR using the forward: 5’-AGTAACAGGCAGGTCCTTCA-3´and reverse: 5 ’-TGATCTTGCCACAGTCTCCA-3’ primers. The 386 bp PCR products were then digested with TseI restriction enzyme (New England Biolabs) for two hours at 65 °C and analyzed by standard agarose gel electrophoresis.
Cell survival assay
The effect of the different FANCM variants on cell survival was measured with a Sulforhodamine B (SRB) assay.21One-thousand cells were seeded in 96-well plates and treated constantly with DEB or PARPi olaparib at the indicated concentrations until untreated cells reached confluency. Cell monolayers werefixed overnight at 4 °C with 75 μl of 20% trichloroacetic acid (TCA). TCA was aspirated, and cells washed with tap water. Once dried, 50μl of SRB was added to the wells and plates were incubated on a shaker at room temperature for 30 min. The excess of SRB dye was removed by washing repeatedly with 1% acetic acid, the plates were dried for 20 min, and the protein-bound dye was dissolved in 10 mM Tris for OD determination at 492 nm using a microplate reader (Tecan Sunrise™,
Tecan Group Ltd. Männedorf, Switzerland). At least three independent experiments were performed for each cell line and in each experiment, 12 wells were measured per concentration point. These results were statistically analyzed using the Prism (GraphPad) software. Two-Way ANOVA test was used for single comparisons between different cell lines and statistical significance was assessed with the Tukey’s range test. A P-value < 0.05 was considered statistically significant.
Chromosome fragility test
Chromosome fragility test was performed as previously described.11 Twenty-five metaphases were scored for chromosome breakages using the Metafer Slide Scanning Platform from Metasystems. Results were graphed as distributions of metaphases presenting 0, 1, and >1 chromatid break. Statistical analysis was performed applying chi-squared test.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary
DATA AVAILABILITY
A subset of the genotype data analysed in this study is publicly available from the dbGaP repository and can be accessed athttps://identifiers.org/dbgap:phs001265.v1.
p1(data generated as part of the BCAC studies) and athttps://identifiers.org/dbgap: phs001321.v1.p1(data generated as part of the CIMBA studies). The remaining genotype data analysed in this study (and generated as part of the BCAC and CIMBA studies listed in Supplementary Tables 4 and 5 of the related article, respectively) are not publicly available due to restraints imposed by the ethics committees of individual studies, but can be accessed from the corresponding author on reasonable request as described at https://doi.org/10.6084/m9.figshare.8982296.22
Additional datasets generated during this study (and supporting Fig. 1 and Supplementary Tables 2 and 3 in the published article) are available on request as described above. The data generated and analyzed during this study are described in the following data record:https://doi.org/10.6084/m9.figshare.8982296.22
Received: 29 March 2019; Accepted: 30 August 2019;
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ACKNOWLEDGEMENTS
Peterlongo laboratory is supported by Associazione Italiana Ricerca sul Cancro (AIRC; IG2015 no.16732) to P. Peterlongo and by a fellowship from Fondazione Umberto Veronesi to G. Figlioli. Surrallés laboratory is supported by the ICREA-Academia program, the Spanish Ministry of Health (projects FANCOSTEM and FANCOLEN), the Spanish Ministry of Economy and Competiveness (projects CB06/07/0023 and RTI2018-098419-B-I00), the European Commission (EUROFANCOLEN project HEALTH-F5-2012-305421 and P-SPHERE COFUND project), the Fanconi Anemia Research Fund Inc, and the“Fondo Europeo de Desarrollo Regional, una manera de hacer Europa” (FEDER). CIBERER is an initiative of the Instituto de Salud Carlos III, Spain. BCAC: we thank all the individuals who took part in these studies and all the researchers, clinicians, technicians and administrative staff who have enabled this work to be carried out. ABCFS thank Maggie Angelakos, Judi Maskiell, Tu Nguyen-Dumont is a National Breast Cancer Foundation (Australia) Career Development Fellow. ABCS thanks the Blood bank Sanquin, The Netherlands. Samples are made available to researchers on a non-exclusive basis. BCEES thanks Allyson Thomson, Christobel Saunders, Terry Slevin, BreastScreen Western Australia, Elizabeth Wylie, Rachel Lloyd. The BCINIS study would not have been possible without the contributions of Dr. Hedy Rennert, Dr. K. Landsman, Dr. N. Gronich, Dr. A. Flugelman, Dr. W. Saliba, Dr. E. Liani, Dr. I. Cohen, Dr. S. Kalet, Dr. V. Friedman, Dr. O. Barnet of the NICCC in Haifa, and all the contributing family medicine, surgery, pathology and oncology teams in all medical institutes in Northern Israel. The BREOGAN study would not have been possible without the contributions of the following: Manuela Gago-Dominguez, Jose Esteban Castelao, Angel Carracedo, Victor Muñoz Garzón, Alejandro Novo Domínguez, Maria Elena Martinez, Sara Miranda Ponte, Carmen Redondo Marey, Maite Peña Fernández, Manuel Enguix Castelo, Maria Torres, Manuel Calaza (BREOGAN), José Antúnez, Máximo Fraga and the staff of the Department of Pathology and Biobank of the University Hospital Complex of Santiago-CHUS, Instituto de Investigación Sanitaria de Santiago, IDIS, Xerencia de Xestion Integrada de Santiago-SERGAS; Joaquín González-Carreró and the staff of the Department of Pathology and Biobank of University Hospital Complex of Vigo, Instituto de Investigacion Biomedica Galicia Sur, SERGAS, Vigo, Spain. BSUCH thanks Peter Bugert, Medical Faculty Mannheim. CBCS thanks study participants, co-investigators, collaborators and staff of the Canadian Breast Cancer Study, and project coordinators Agnes Lai and Celine Morissette. CCGP thanks Styliani Apostolaki, Anna Margiolaki, Georgios Nintos, Maria Perraki, Georgia Saloustrou, Georgia Sevastaki, Konstantinos Pompodakis. CGPS thanks staff and participants of the Copenhagen General Population Study. For the excellent technical assistance: Dorthe Uldall Andersen, Maria Birna Arnadottir, Anne Bank, Dorthe Kjeldgård Hansen. The Danish Cancer Biobank is acknowledged for providing infrastructure for the collection of blood samples for the cases. Investigators from the CPS-II cohort thank the participants and Study Management Group for their invaluable contributions to this research. They also acknowledge the contribution to this study from central cancer registries supported through the Centers for Disease Control and Prevention National Program of Cancer Registries, as well as cancer registries supported by the National Cancer Institute Surveillance Epidemiology and End Results program. The CTS Steering Committee includes Leslie Bernstein, Susan Neuhausen, James Lacey, Sophia Wang, Huiyan Ma, and Jessica Clague DeHart at the Beckman Research Institute of City of Hope, Dennis Deapen, Rich Pinder, and Eunjung Lee at the University of Southern California, Pam Horn-Ross, Peggy Reynolds, Christina Clarke Dur and David Nelson at the Cancer Prevention Institute of California, Hoda Anton-Culver, Argyrios Ziogas, and
Hannah Park at the University of California Irvine, and Fred Schumacher at Case Western University. DIETCOMPLYF thanks the patients, nurses and clinical staff involved in the study. The DietCompLyf study was funded by the charity Against Breast Cancer (Registered Charity Number 1121258) and the NCRN. We thank the participants and the investigators of EPIC (European Prospective Investigation into Cancer and Nutrition). ESTHER thanks Hartwig Ziegler, Sonja Wolf, Volker Hermann, Christa Stegmaier, Katja Butterbach. FHRISK thanks NIHR for funding. GC-HBOC thanks Stefanie Engert, Heide Hellebrand, Sandra Kröber and LIFE - Leipzig Research Centre for Civilization Diseases (Markus Loeffler, Joachim Thiery, Matthias Nüchter, Ronny Baber). The GENICA Network: Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, and University of Tübingen, Germany [HB, Wing-Yee Lo], German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ) [HB], Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy - EXC 2180 - 390900677 [HB], Department of Internal Medicine, Evangelische Kliniken Bonn gGmbH, Johanniter Krankenhaus, Bonn, Germany [Yon-Dschun Ko, Christian Baisch], Institute of Pathology, University of Bonn, Germany [Hans-Peter Fischer], Molecular Genetics of Breast Cancer, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany [Ute Hamann], Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr University Bochum (IPA), Bochum, Germany [TB, Beate Pesch, Sylvia Rabstein, Anne Lotz]; and Institute of Occupational Medicine and Maritime Medicine, University Medical Center Hamburg-Eppendorf, Germany [Volker Harth]. HABCS thanks Michael Bremer. HEBCS thanks Heidi Toiminen, Kristiina Aittomäki, Irja Erkkilä and Outi Malkavaara. HMBCS thanks Peter Hillemanns, Hans Christiansen and Johann H. Karstens. HUBCS thanks Shamil Gantsev. KARMA thanks the Swedish Medical Research Counsel. KBCP thanks Eija Myöhänen, Helena Kemiläinen. LMBC thanks Gilian Peuteman, Thomas Van Brussel, EvyVanderheyden and Kathleen Corthouts. MABCS thanks Milena Jakimovska (RCGEB“Georgi D. Efremov), Katerina Kubelka, Mitko Karadjozov (Adzibadem-Sistina” Hospital), Andrej Arsovski and Liljana Stojanovska (Re-Medika” Hospital) for their contributions and commitment to this study. MARIE thanks Petra Seibold, Dieter Flesch-Janys, Judith Heinz, Nadia Obi, Alina Vrieling, Sabine Behrens, Ursula Eilber, Muhabbet Celik, Til Olchers and Stefan Nickels. MBCSG (Milan Breast Cancer Study Group) thanks Daniela Zaffaroni, Irene Feroce, and the personnel of the Cogentech Cancer Genetic Test Laboratory. We thank the coordinators, the research staff and especially the MMHS participants for their continued collaboration on research studies in breast cancer. MSKCC thanks Marina Corines and Lauren Jacobs. MTLGEBCS would like to thank Martine Tranchant (CHU de Québec Research Center), Marie-France Valois, Annie Turgeon and Lea Heguy (McGill University Health Center, Royal Victoria Hospital; McGill University) for DNA extraction, sample management and skillful technical assistance. J.S. is Chairholder of the Canada Research Chair in Oncogenetics. NBHS thanks study participants and research staff for their contributions and commitment to the studies. We would like to thank the participants and staff of the Nurses’ Health Study and Nurses’ Health Study II for their valuable contributions as well as the following state cancer registries for their help: AL, AZ, AR, CA, CO, CT, DE, FL, GA, ID, IL, IN, IA, KY, LA, ME, MD, MA, MI, NE, NH, NJ, NY, NC, ND, OH, OK, OR, PA, RI, SC, TN, TX, VA, WA, WY. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health, and those of participating registries as required. The authors assume full responsibility for analyses and interpretation of these data. OFBCR thanks Teresa Selander and Nayana Weerasooriya. ORIGO thanks E. Krol-Warmerdam, and J. Blom for patient accrual, administering questionnaires, and managing clinical information. PBCS thanks Louise Brinton, Mark Sherman, Neonila Szeszenia-Dabrowska, Beata Peplonska, Witold Zatonski, Pei Chao and Michael Stagner. The ethical approval for the POSH study is MREC /00/6/69, UKCRN ID: 1137. We thank staff in the Experimental Cancer Medicine Centre (ECMC) supported Faculty of Medicine Tissue Bank and the Faculty of Medicine DNA Banking resource. PREFACE thanks Sonja Oeser and Silke Landrith. PROCAS thanks NIHR for funding. RBCS thanks Petra Bos, Jannet Blom, Ellen Crepin, Elisabeth Huijskens, Anja Kromwijk-Nieuwlaat, Annette Heemskerk, the Erasmus MC Family Cancer Clinic. We thank the SEARCH and EPIC teams. SKKDKFZS thanks all study participants, clinicians, family doctors, researchers and technicians for their contributions and commitment to this study. We thank the SUCCESS Study teams in Munich, Duessldorf, Erlangen and Ulm. SZBCS thanks Ewa Putresza. UCIBCS thanks Irene Masunaka. UKBGS thanks Breast Cancer Now and the Institute of Cancer Research for support and funding of the Breakthrough Generations Study, and the study participants, study staff, and the doctors, nurses and other health care providers and health information sources who have contributed to the study. We acknowledge NHS funding to the Royal Marsden/ICR NIHR Biomedical Research Centre. CIMBA: we are grateful to all the families and clinicians who contribute to the studies; Sue Healey, in particular taking on the task of mutation classification with the late Olga Sinilnikova; Maggie Angelakos, Judi Maskiell, Helen Tsimiklis; members and participants in the New York site of the Breast Cancer Family Registry; members and participants in the Ontario Familial Breast Cancer Registry; Vilius Rudaitis and Laimonas Griškevičius; Yuan Chun Ding and Linda Steele for their work in participant enrollment and biospecimen and data management; Bent Ejlertsen and Anne-Marie
Gerdes for the recruitment and genetic counseling of participants; Alicia Barroso, Rosario Alonso and Guillermo Pita; all the individuals and the researchers who took part in CONSIT TEAM (Consorzio Italiano Tumori Ereditari Alla Mammella), thanks in particular: Giulia Cagnoli, Roberta Villa, Irene Feroce, Mariarosaria Calvello, Riccardo Dolcetti, Giuseppe Giannini, Laura Papi, Gabriele Lorenzo Capone, Liliana Varesco, Viviana Gismondi, Maria Grazia Tibiletti, Daniela Furlan, Antonella Savarese, Aline Martayan, Stefania Tommasi, Brunella Pilato, Isabella Marchi, Elena Bandieri, Antonio Russo, Daniele Calistri and the personnel of the Cogentech Cancer Genetic Test Laboratory, Milan, Italy. FPGMX: members of the Cancer Genetics group (IDIS): Ana Blanco, Miguel Aguado, Uxía Esperón and Belinda Rodríguez. We thank all participants, clinicians, family doctors, researchers, and technicians for their contributions and commitment to the DKFZ study and the collaborating groups in Lahore, Pakistan (Noor Muhammad, Sidra Gull, Seerat Bajwa, Faiz Ali Khan, Humaira Naeemi, Saima Faisal, Asif Loya, Mohammed Aasim Yusuf) and Bogota, Colombia (Diana Torres, Ignacio Briceno, Fabian Gil). Genetic Modifiers of Cancer Risk in BRCA1/ 2 Mutation Carriers (GEMO) study is a study from the National Cancer Genetics Network UNICANCER Genetic Group, France. We wish to pay a tribute to Olga M. Sinilnikova, who with Dominique Stoppa-Lyonnet initiated and coordinated GEMO until she sadly passed away on the 30th June 2014. The team in Lyon (Olga Sinilnikova, Mélanie Léoné, Laure Barjhoux, Carole Verny-Pierre, Sylvie Mazoyer, Francesca Damiola, Valérie Sornin) managed the GEMO samples until the biological resource centre was transferred to Paris in December 2015 (Noura Mebirouk, Fabienne Lesueur, Dominique Stoppa-Lyonnet). We want to thank all the GEMO collaborating groups for their contribution to this study. Drs.Sofia Khan, Irja Erkkilä and Virpi Palola; The Hereditary Breast and Ovarian Cancer Research Group Netherlands (HEBON) consists of the following Collaborating Centers: Netherlands Cancer Institute (coordinating center), Amsterdam, NL: M.A. Rookus, F.B.L. Hogervorst, F.E. van Leeuwen, M.A. Adank, M.K. Schmidt, N.S. Russell, D.J. Jenner; Erasmus Medical Center, Rotterdam, NL: J.M. Collée, A.M.W. van den Ouweland, M.J. Hooning, C.M. Seynaeve, C.H.M. van Deurzen, I.M. Obdeijn; Leiden University Medical Center, NL: C.J. van Asperen, P. Devilee, T.C.T.E.F. van Cronenburg; Radboud University Nijmegen Medical Center, NL: C.M. Kets, A.R. Mensenkamp; University Medical Center Utrecht, NL: M.G.E.M. Ausems, M.J. Koudijs; Amsterdam Medical Center, NL: C.M. Aalfs, H.E.J. Meijers-Heijboer; VU University Medical Center, Amsterdam, NL: K. van Engelen, J.J.P. Gille; Maastricht University Medical Center, NL: E.B. Gómez-Garcia, M.J. Blok; University of Groningen, NL: J.C. Oosterwijk, A.H. van der Hout, M.J. Mourits, G.H. de Bock; The Netherlands Comprehensive Cancer Organisation (IKNL): S. Siesling, J. Verloop; The nationwide network and registry of histo- and cytopathology in The Netherlands (PALGA): A.W. van den Belt-Dusebout. HEBON thanks the study participants and the registration teams of IKNL and PALGA for part of the data collection. Overbeek; the Hungarian Breast and Ovarian Cancer Study Group members (Janos Papp, Aniko Bozsik, Zoltan Matrai, Miklos Kasler, Judit Franko, Maria Balogh, Gabriella Domokos, Judit Ferenczi, Department of Molecular Genetics, National Institute of Oncology, Budapest, Hungary) and the clinicians and patients for their contributions to this study; HVH (University Hospital Vall d’Hebron) the authors acknowledge the Oncogenetics Group (VHIO) and the High Risk and Cancer Prevention Unit of the University Hospital Vall d’Hebron, Miguel Servet Progam (CP10/00617), and the Cellex Foundation for providing research facilities and equipment; the ICO Hereditary Cancer Program team led by Dr. Gabriel Capella; the ICO Hereditary Cancer Program team led by Dr. Gabriel Capella; Dr Martine Dumont for sample management and skillful assistance; Catarina Santos and Pedro Pinto; members of the Center of Molecular Diagnosis, Oncogenetics Department and Molecular Oncology Research Center of Barretos Cancer Hospital; Heather Thorne, Eveline Niedermayr, all the kConFab investigators, research nurses and staff, the heads and staff of the Family Cancer Clinics, and the Clinical Follow Up Study (which has received funding from the NHMRC, the National Breast Cancer Foundation, Cancer Australia, and the National Institute of Health (USA)) for their contributions to this resource, and the many families who contribute to kConFab; the investigators of the Australia New Zealand NRG Oncology group; members and participants in the Ontario Cancer Genetics Network; Kevin Sweet, Caroline Craven, Julia Cooper, Amber Aielts, and Michelle O’Conor; Christina Selkirk; Helena Jernström, Karin Henriksson, Katja Harbst, Maria Soller, Ulf Kristoffersson; from Gothenburg Sahlgrenska University Hospital: Anna Öfverholm, Margareta Nordling, Per Karlsson, Zakaria Einbeigi; from Stockholm and Karolinska University Hospital: Anna von Wachenfeldt, Annelie Liljegren, Annika Lindblom, Brita Arver, Gisela Barbany Bustinza; from Umeå University Hospital: Beatrice Melin, Christina Edwinsdotter Ardnor, Monica Ema-nuelsson; from Uppsala University: Hans Ehrencrona, Maritta Hellström Pigg, Richard Rosenquist; from Linköping University Hospital: Marie Stenmark-Askmalm, Sigrun Liedgren; Cecilia Zvocec, Qun Niu; Joyce Seldon and Lorna Kwan; Dr. Robert Nussbaum, Beth Crawford, Kate Loranger, Julie Mak, Nicola Stewart, Robin Lee, Amie Blanco and Peggy Conrad and Salina Chan; Carole Pye, Patricia Harrington and Eva Wozniak. OSUCCG thanks Kevin Sweet, Caroline Craven, Julia Cooper, Michelle O’Conor and Amber Aeilts. BCAC is funded by Cancer Research UK [C1287/A16563, C1287/A10118], the European Union’s Horizon 2020 Research and Innovation Programme (grant numbers 634935 and 633784 for BRIDGES and B-CAST
respectively), and by the European Community´s Seventh Framework Programme under grant agreement number 223175 (grant number HEALTH-F2-2009-223175) (COGS). The EU Horizon 2020 Research and Innovation Programme funding source had no role in study design, data collection, data analysis, data interpretation or writing of the report. Genotyping of the OncoArray was funded by the NIH Grant U19 CA148065, and Cancer UK Grant C1287/A16563 and the PERSPECTIVE project supported by the Government of Canada through Genome Canada and the Canadian Institutes of Health Research (grant GPH-129344) and, the Ministère de l’Économie, Science et Innovation du Québec through Genome Québec and the PSRSIIRI-701 grant, and the Quebec Breast Cancer Foundation. The Australian Breast Cancer Family Study (ABCFS) was supported by grant UM1 CA164920 from the National Cancer Institute (USA). The content of this manuscript does not necessarily reflect the views or policies of the National Cancer Institute or any of the collaborating centers in the Breast Cancer Family Registry (BCFR), nor does mention of trade names, commercial products, or organizations imply endorsement by the USA Government or the BCFR. The ABCFS was also supported by the National Health and Medical Research Council of Australia, the New South Wales Cancer Council, the Victorian Health Promotion Foundation (Australia) and the Victorian Breast Cancer Research Consortium. J.L.H. is a National Health and Medical Research Council (NHMRC) Senior Principal Research Fellow. M.C.S. is a NHMRC Senior Research Fellow. The ABCS study was supported by the Dutch Cancer Society [grants NKI 2007-3839; 2009 4363]. The Australian Breast Cancer Tissue Bank (ABCTB) was supported by the National Health and Medical Research Council of Australia, The Cancer Institute NSW and the National Breast Cancer Foundation. The AHS study is supported by the intramural research program of the National Institutes of Health, the National Cancer Institute (grant number Z01-CP010119), and the National Institute of Environmental Health Sciences (grant number Z01-ES049030). The work of the BBCC was partly funded by ELAN-Fond of the University Hospital of Erlangen. The BBCS is funded by Cancer Research UK and Breast Cancer Now and acknowledges NHS funding to the NIHR Biomedical Research Centre, and the National Cancer Research Network (NCRN). The BCEES was funded by the National Health and Medical Research Council, Australia and the Cancer Council Western Australia. For the BCFR-NY, BCFR-PA, BCFR-UT this work was supported by grant UM1 CA164920 from the National Cancer Institute. The content of this manuscript does not necessarily reflect the views or policies of the National Cancer Institute or any of the collaborating centers in the Breast Cancer Family Registry (BCFR), nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government or the BCFR. BCINIS study was funded by the BCRF (The Breast Cancer Research Foundation, USA). The BREast Oncology GAlician Network (BREOGAN) is funded by Acción Estratégica de Salud del Instituto de Salud Carlos III FIS PI12/02125/Cofinanciado FEDER; Acción Estratégica de Salud del Instituto de Salud Carlos III FIS Intrasalud (PI13/01136); Programa Grupos Emergentes, Cancer Genetics Unit, Instituto de Investigacion Biomedica Galicia Sur. Xerencia de Xestion Integrada de Vigo-SERGAS, Instituto de Salud Carlos III, Spain; Grant 10CSA012E, Consellería de Industria Programa Sectorial de Investigación Aplicada, PEME I+ D e I + D Suma del Plan Gallego de Investigación, Desarrollo e Innovación Tecnológica de la Consellería de Industria de la Xunta de Galicia, Spain; Grant EC11-192. Fomento de la Investigación Clínica Independiente, Ministerio de Sanidad, Servicios Sociales e Igualdad, Spain; and Grant FEDER-Innterconecta. Ministerio de Economia y Competitividad, Xunta de Galicia, Spain. The BSUCH study was supported by the Dietmar-Hopp Foundation, the Helmholtz Society and the German Cancer Research Center (DKFZ). Sample collection and processing was funded in part by grants from the National Cancer Institute (NCI R01CA120120 and K24CA169004). CBCS is funded by the Canadian Cancer Society (grant # 313404) and the Canadian Institutes of Health Research. CCGP is supported by funding from the University of Crete. The CECILE study was supported by Fondation de France, Institut National du Cancer (INCa), Ligue Nationale contre le Cancer, Agence Nationale de Sécurité Sanitaire, de l’Alimentation, de l’Environnement et du Travail (ANSES), Agence Nationale de la Recherche (ANR). The CGPS was supported by the Chief Physician Johan Boserup and Lise Boserup Fund, the Danish Medical Research Council, and Herlev and Gentofte Hospital. The American Cancer Society funds the creation, maintenance, and updating of the CPS-II cohort. The CTS was initially supported by the California Breast Cancer Act of 1993 and the California Breast Cancer Research Fund (contract 97-10500) and is currently funded through the National Institutes of Health (R01 CA77398, K05 CA136967, UM1 CA164917, and U01 CA199277). Collection of cancer incidence data was supported by the California Department of Public Health as part of the statewide cancer reporting program mandated by California Health and Safety Code Section 103885. The University of Westminster curates the DietCompLyf database funded by Against Breast Cancer Registered Charity No. 1121258 and the NCRN. The coordination of EPIC isfinancially supported by the European Commission (DG-SANCO) and the International Agency for Research on Cancer. The national cohorts are supported by: Ligue Contre le Cancer, Institut Gustave Roussy, Mutuelle Générale de l’Education Nationale, Institut National de la Santé et de la Recherche Médicale (INSERM) (France); German Cancer Aid, German Cancer Research Center (DKFZ), Federal Ministry of Education and Research (BMBF) (Germany); the Hellenic Health Foundation, the Stavros Niarchos
